Granzyme A and granzyme B diagnostics

ABSTRACT

A method for identifying a subject being at risk for or having a chronic inflammatory disease, fibrillinopathy, atherosclerosis, or coronary artery disease is provided. The method may include determining the concentration of GrA and/or GrB in a blood or serum sample from said subject; and comparing the concentrations to the corresponding concentration in a control sample, wherein an elevated concentration of GrA and/or GrB may be indicative of a chronic inflammatory disease, fibrillinopathy, atherosclerosis, or coronary artery disease. The method may further include identifying concentrations of fibrinogen, elastin and/or fibrillin.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/867,870, filed Apr. 22, 2013, now U.S. Pat. No. 8,715,948, which is adivision of U.S. patent application Ser. No. 12/681,349, filed Aug. 31,2010, now U.S. Pat. No. 8,426,149, which is a National Stage ofInternational Application No. PCT/CA2008/001752, filed Oct. 1, 2008,which claims the benefit of U.S. Provisional Application No. 60/996,139,filed Nov. 2, 2007; U.S. Provisional Application No. 60/996,138, filedNov. 2, 2007; and U.S. Provisional Application No. 60/960,478, filedOct. 1, 2007, each expressly incorporated herein by reference in itsentirety

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 43586_SEQ_Final_(—)2014-04-23.txt. The text fileis 2 KB; was created on Apr. 23, 2014; and is being submitted viaEFS-Web with the filing of the specification.

TECHNICAL FIELD

This invention relates to the field of diagnostics. More particularly,to the diagnosis of diseases and conditions by measuring granzyme Blevels and/or granzyme A levels.

BACKGROUND

The granzymes are highly conserved group of serine proteases, with fivemembers (A, B, H, K and M) in humans and ten members (A-G, K, M-N) inmice (Sattar R. et al., Biochem Biophys Res Commun 308:726-35 (2003)).Granzyme B (GrB or cytotoxic T-lymphocyte (CTL)-associated genetranscript-l-Brunet J. F. et al., Nature 322:268-71 (1986)), has beenreported as being involved in anti-viral and anti-tumour functions, andis associated with autoimmunity, transplant rejection, graft-versus-hostdisease, and thymocyte development (Barry M. & Bleackley R. C., Nat RevImmunol 2:401-9 (2002)). Granzyme A (GrA) is also involved inimmune-mediated killing, and is expressed by both innate and adaptiveimmune cytotoxic cells.

GrB is reported to have a contribution to CTL-mediated target cellapoptosis. GrB-deficient mice possess a normal phenotype, with theexception of a slightly reduced CTL-mediated target cell apoptosis,anti-viral responses and tumor cell clearance (Revell P. A. et al., JImmunol 174:2124-31 (2005); and Heusel J. W. et al., Cell 76:977-87(1994)), suggesting a redundancy in immune mediated cell removal.GrB-deficient recipient mice exhibit reduced allograft vasculopathy(Choy J. C. et al., Am J Transplant 5:494-9 (2005)), and its deficiencyin mice leads to increased susceptibility to allergen-induced asthma(Devadas S. et al., Immunity 25:237-47 (2006)).

Buzza M. S. et al. report that plasma GrA levels in normal individualsare between 15-35 pg/ml and plasma GrB levels in normal individuals areup to 15 pg/ml (Biol. Chem. 387:827-837 (2006)). Skjelland et al. teachthat plasma levels of granzyme B are increased in patients with lipidrich carotid plaques, but also teach that normal plasma levels of GrBcan be up to 100 pg/ml, while patterns with unstable plaques have plasmalevels of GrB over about 100 pg/ml (and up to about 650 pg/ml) andpatients with stable plaques have plasma levels of GrB between about 25pg/ml and about 400 pg/ml (Atherosclerosis 195:e142-e146 (2007)).Furthermore, GrB levels have been measured in the supernatant ofcultured peripheral blood mononuclear cells isolated from patients atabout 40 pg/ml for patients with unstable angina pectoris and at about18 pg/ml for patients with stable angina pectoris (Tsuru R. et al.,Heart 94:305-310 (2008) e-published Jun. 25, 2007). GrB has also beenmeasured in rheumatoid arthritis patients (Goldbach-Mansky et al., AnnRheum Dis. 64:715-721 (2005); Kraan et al., Ann Rheum Dis 63:483-488(2004); Villanueva et al., Arthritis Res Ther 7:R30-R37 (2005)). Choy J.C. et al. reported increased levels of GrB in patients with advancedatherosclerosis (Mod Pathol 16:460-70 (2003)). GrB has also beenassociated with aortic aneurisms and with atherosclerosis plaquedestabilization (Choy et al. Arterioscler. Thromb. Vasc. Biol.24:2245-2250, (2004)). Kim et al., show that macrophages expressgranzyme B in the lesion areas of atherosclerosis and rheumatoidarthritis (Immunology Letters 111:57-65 (2007)). Increased GrB levels inChronic Obstructive Pulmonary Disease (COPD) patients were reported inbronchoalveolar lavage (BAL) derived T-cells (Hodge et al., J. of COPD3:179-187 (2006)). Also GrB produced protein fragments are reported inSjögren's Syndrome patients (Huang et al., Clin Exp Immun 142:148-154(2005)). Additionally, GrA and GrB are reported in BAL fluids frompatients with inflammatory lung disease (Tremblay et al., J Immunology165:3966-3969 (2000)). Thewissen et al. compares GrA and GrB levels inrheumatoid arthritis (RA), multiple sclerosis (MS), and between healthyindividuals, and reports no change in GrA levels between healthypatients and RA or MS patients for GrA and reports a decrease in GrBlevels for MS patients relative to healthy patients (Clinical Immunology123:209-218 (2007)). GrA released in the brain may be associated withautoimmune disorders of the nervous system (Suidan et al., PNAS91:8112-8116 (1994)). Vernooy et al. report increased GrA expression intype II pneumocytes of patients with severe COPD (Am J Respir Crit CareMed 175:464-472 (2007)). Immunodiagnostic methods for Granzymes A and Bare also known (for example WO 99/54737).

SUMMARY

This invention is based, in part, on the discovery of the contributiongranzyme A makes to certain diseases, including chronic inflammatorydiseases, atherosclerosis or coronary artery disease, and chronicobstructive pulmonary disease (COPD), and in particular to theextracellular activity of granzyme A whereby granzyme A cleavesextracellular matrix proteins such as fibronectin.

This invention is also based, in part, on the discovery of thecontribution granzyme B makes to certain diseases, including chronicinflammatory diseases, atherosclerosis or coronary artery disease, andchronic obstructive pulmonary disease (COPD), and in particular to theextracellular activity of granzyme B whereby granzyme B cleavesextracellular matrix proteins such as elastin, Fibulin-2, Fibrillin-1,and Fibrillin-2.

In one aspect of the present invention, there is provided a method fordiagnosis of chronic inflammatory disease in a subject suspected ofhaving chronic inflammatory disease or having chronic inflammatorydisease, the method including: determining the concentration of GrA in ablood plasma or serum sample from the subject; and comparing theconcentrations to the corresponding concentration in a control sample,wherein an elevated concentration of GrA is indicative of chronicinflammatory disease.

In a further aspect of the present invention, there is provided a methodfor diagnosis of atherosclerosis or coronary artery disease in a subjectsuspected of having atherosclerosis or coronary artery disease or havingatherosclerosis or coronary artery disease, the method including:determining the concentration of GrA in a blood plasma or serum samplefrom the subject; and comparing the concentrations to the correspondingconcentration in a control sample, wherein an elevated concentration ofGrA is indicative of atherosclerosis or coronary artery disease.

In a further aspect of the present invention, there is provided a methodfor diagnosis of a fibrillinopathy in a subject suspected of havingfibrillinopathy or having fibrillinopathy, the method including:determining the concentration of GrA in a blood plasma or serum samplefrom the subject; and comparing the concentrations to the correspondingconcentration in a control sample, wherein an elevated concentration ofGrA is indicative of fibrillinopathy.

The method may further include determining the concentration of one ormore of: fibronectin; and fibrillin; with reference to the controlsample as indicative of chronic inflammatory disease. The concentrationof GrA, fibronectin and/or fibrillin may be determined by animmunodiagnostic assay. The immunodiagnostic assay may be anenzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbentspot (ELISPOT), dot blot, Western blot, or other proteomics assay, etc.The subject may have a GrA blood plasma concentration >20 pg/ml and/or afibronectin blood plasma concentration >400 μg/ml as an indication ofchronic inflammatory disease, atherosclerosis or coronary arterydisease, or fibrillinopathy. The fibronectin or fibrillin, may be afibronectin degradation product or an fibrillin degradation product. Themethod may further include one or more of: diagnostic imaging; clinicaldiagnosis and alternative laboratory diagnostics.

The chronic inflammatory disease may be selected from one or more of:rheumatoid arthritis; osteoarthritis; inflammatory bowel disease;psoriasis; lupus crythematosus; multiple sclerosis; Sjogren's syndrome;polymyositis; dermatomyositis; vasculitis; asthma and mixed connectivetissue disease.

The fibrillinopathy may be selected from one or more of: Marfansyndrome; Beal's syndrome; congenital contractural arachnactyly;supravalvular aortic stenosis; Williams-Beuren syndrome; autosomalrecessive cutis laxa; autosomal dominant cutis laxa; and acquired cutislaxa.

In a further aspect of the present invention, there is provided a methodfor diagnosis of chronic inflammatory disease in a subject suspected ofhaving chronic inflammatory disease or having chronic inflammatorydisease, the method including: determining the concentration of GrB in ablood plasma or serum sample from the subject; and comparing theconcentrations to the corresponding concentration in a control sample,wherein an elevated concentration of GrB is indicative of chronicinflammatory disease.

In a further aspect of the present invention, there is provided a methodfor diagnosis of a fibrillinopathy in a subject suspected of having afibrillinopathy or having a fibrillinopathy, the method including:determining the concentration of GrB in a blood plasma or serum samplefrom the subject; and comparing the concentrations to the correspondingconcentration in a control sample, wherein an elevated concentration ofGrB is indicative of fibrillinopathy.

The method may further include determining the concentration of one ormore of: elastin; Fibronectin; Fibulin-2; Fibrillin-1; and Fibrillin-2;with reference to the control sample as indicative of chronicinflammatory disease. The concentration of GrB or elastin or fibrillinor fibulin or fibronectin may be determined by an immunodiagnosticassay. The immunodiagnostic assay may be an enzyme-linked Immunosorbentassay (ELISA), enzyme-linked immunosorbent spot (ELISPOT), dot blot,Western blot, or other proteomics assay, etc. The subject may have a GrBblood plasma concentration >40 pg/ml and/or a fibronectin blood plasmaconcentration >400 μg/ml as an indication of chronic inflammatorydisease, or fibrillinopathy. The elastin or fibrillin or fibulin orfibronectin, may be an elastin degradation product or a fibrillindegradation product or fibulin degradation product or a fibronectindegradation product. The method may further include one or more of:diagnostic imaging; clinical diagnosis and alternative laboratorydiagnostics.

The chronic inflammatory disease is selected from one or more of:osteoarthritis: inflammatory bowel disease; psoriasis; lupuserythematosus: scleroderma; multiple sclerosis; polymyositis;dermatomyositis; vasculitis; asthma; and mixed connective tissuedisease.

The fibrillinopathy is selected from one or more of Marfan syndrome;Beal's syndrome; congenital contractural arachnactyly; supravalvularaortic stenosis; Williams-Beuren syndrome; autosomal recessive cutislaxa; autosomal dominant cutis laxa; and acquired cutis laxa.

The chronic inflammatory disease may be arthritis. The chronicinflammatory disease may be asthma. The sample from the subject and thenormal sample may be blood plasma samples or blood serum samples. Thesample from the subject and the normal sample may bebronchoalveolarlavages.

A GrA concentration greater than about 20 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrA concentration greater thanabout 21 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrA concentration greater than about 22 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrA concentration greater thanabout 23 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrA concentration greater than about 24 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrA concentration greater thanabout 25 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrA concentration greater than about 30 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrA concentration greater thanabout 35 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrA concentration greater than about 40 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrA concentration greater thanabout 45 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrA concentration greater than about 50 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy.

A GrB concentration greater than about 40 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrB concentration greater thanabout 41 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrB concentration greater than about 42 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrB concentration greater thanabout 43 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrB concentration greater than about 44 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrB concentration greater thanabout 45 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrB concentration greater than about 50 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrB concentration greater thanabout 55 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrB concentration greater than about 60 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrB concentration greater thanabout 65 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrB concentration greater than about 70 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrB concentration greater thanabout 75 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrB concentration greater than about 80 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy. A GrB concentration greater thanabout 90 pg/ml may be considered indicative of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.A GrB concentration greater than about 100 pg/ml may be consideredindicative of chronic inflammatory disease, atherosclerosis or coronaryartery disease, or fibrillinopathy.

A fibronectin blood plasma concentration >400 μg/ml may be an indicationof chronic inflammatory disease, atherosclerosis or coronary arterydisease, or fibrillinopathy either alone or in combination with a GrAand/or GrB concentration as set out herein. A fibronectin blood plasmaconcentration >450 μg/ml may be an indication of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathyeither alone or in combination with a GrA and/or GrB concentration asset out herein. A fibronectin blood plasma concentration >500 μg/ml maybe an indication of chronic inflammatory disease, atherosclerosis orcoronary artery disease, or fibrillinopathy either alone or incombination with a GrA and/or GrB concentration as set out herein.Furthermore, concentrations of elastin, Fibulin-2, Fibrillin-1, and/orFibrillin-2 or degradation products thereof may be indicative of chronicinflammatory disease, atherosclerosis or coronary artery disease, orfibrillinopathy in a subject. Alternatively, the methods describedherein may also be used to make a diagnosis of COPD in a subject.

In a further aspect of the present invention, there are provided kits,commercial packages and uses for the diagnosis of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathy.The kits and commercial packages may also include one or more of:reagents, antibodies, normal controls, a listing of normal levels andthose associated with a diagnosis of one or more of chronic inflammatorydisease, atherosclerosis or coronary artery disease, or fibrillinopathyor COPD, and/or instructions for their use. The methods may also be usedin conjunction with known diagnostic methods.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the average calculated LDL-C in the plasmaof C57/Bl/6, GrB KO, ApoE KO or ApoE/GrB DKO mice. White bars representmice fed a normal chow diet; black bars represent mice fed a Westerndiet. N=3 for each group. Calculated LDL-C (mg/ml) is the Y-axis.

FIG. 2 is a bar graph showing the average total cholesterol in theplasma of C57/Bl/6, GrB KO, ApoE KO or ApoE/GrB DKO mice. White barsrepresent mice fed a normal chow diet; black bars represent mice fed aWestern diet. N=3 for each group. Total cholesterol (mg/ml) is theY-axis.

FIG. 3 is a bar graph showing the plasma lipid profiles of C57/Bl/6(solid bar), GrB KO (white bar), ApoE KO (hatched bar) or ApoE/GrB DKO(checked bar) mice on a Western diet. TG average-average triglycerides:TC average—total cholesterol average. N=3 for each group.

FIG. 4 shows the percentage area of the aortic root in ApoE KO (whitebar) and ApoE/GrB DKO (black bar) mice fed a Western diet. N=2 for theDKO mice, N=4 for the ApoE KO mice. Values for each section wascalculated (sum of the plaque area)/(total aortic root area)*100%. Foreach animal, 3-7 sections of aortic roots were analyzed for % lesionarea and averaged.

FIG. 5 Representative artery sections from mice fed a Western diet for30 weeks. (A) C57 WT, (B) GrB−/−, (C) ApoE−/−, (D) ApoE/GrB-DKO.

FIG. 6 Skin of dehaired mice. C57 BL/6 (wt), GrB −/−, ApoE −/− andGrB/ApoE double knockout mice (DKO) with hair removed show the varyingskin conditions associated with the gene knockout phenotypes.

FIG. 7 Representative skin samples at 30 weeks. Hair follicles aredeeply embedded in both the GrB-KO and GrB/ApoE-DKO. Hair follicledensity is also much greater in the GrB/ApoE DKO. Xanthoma formation isvisible in the ApoE-KO and absent from GrB/ApoE-DKO. There also appearsto be a change in the extracellular matrix composition in the GrB-KO andDKO mice as the white area/processing artifact is always present inthese tissues in the dermis and likely due to changes in hydrophobicityin the matrix and fixation procedures.

FIG. 8 is a bar graph showing the Log of Granzyme B levels (pg/ml) oftwo distinct subject groups: GOLD 1 & 2 are a normal subject group andGOLD 3 & 4 are a subject group having chrome obstructive pulmonarydisease (COPD).

FIG. 9 is a bar graph showing the Log of Granzyme B levels (pg/ml), whenadjusted for age and smoking status of two distinct subject groups; GOLD1 & 2 are a normal subject group and GOLD 3 & 4 are a subject grouphaving COPD.

FIG. 10 is a bar graph comparing granzyme B levels in two subjectgroups: smokers and non-smokers. N=3 for both subject groups.

FIG. 11 granzyme B degrades elastin in vitro. Granzyme B was incubatedwith ³H-elastin for 7 days at room temperature. Elastase was incubatedwith ³H-elastin for 2 hours. Supernatants containing the soluble elastincleavage fragments were collected and counted. Data is represented asfold increase in radioactivity over the control (elastin only). (n=2)

FIG. 12 is a bar graph illustrating that the granzyme A is elevated inthe plasma of patients with confirmed atherosclerosis. Plasma sampleswere obtained from patients with (n=5) or without (n=5) coronary arterydisease (CAD).

FIG. 13 is a bar graph illustrating the that granzyme A is elevated inthe plasma of patients that smoke. Plasma samples were obtained frompatients that do (black bar; n=3) or do not (white bar; n=3) smoke.

FIG. 14A illustrates extracellular proteins visualized by Western blotfor biotin. Increased levels of fragmented extracellular matrix proteinswere observed in granzyme A-treated plates.

FIG. 14B illustrates supernatants treated with granzyme A for theindicated times were probed for fibronectin and several fragments wereobserved, as indicated by the arrows, by Western blot.

DETAILED DESCRIPTION

The methods described herein for identifying a subject being at risk foror having one or more of: a chronic inflammatory disease;fibrillinopathy; atherosclerosis; or coronary artery disease; may bepracticed alone or in combination depending on the result desired andthe subject being tested. The method may include determining theconcentration and/or relative amount of GrA and/or GrB in a blood orserum sample from the subject; and comparing the concentration and/orrelative amount to the corresponding concentration in a control sample,wherein an elevated concentration and/or relative amount of GrA and/orGrB may be indicative of a chronic inflammatory disease,fibrillinopathy, atherosclerosis, or coronary artery disease. The methodmay further include identifying a concentration and/or relative amountof one or more of fibrinogen, elastin and/or fibrillin. Where theconcentration and/or relative amount of GrA is determined it may also beinformative to know the concentration and/or relative amount of one ormore of fibrinogen and/or fibrillin. Where the concentration and/orrelative amount of GrB is determined it may also be informative to knowthe concentration and/or relative amount of one or more of elastinand/or fibrillin. A control sample may be obtained from one or morecontrol subjects. Control subjects do not have any one or more of achronic inflammatory disease; fibrillinopathy; atherosclerosis; orcoronary artery disease.

When the concentration and/or relative amount of GrA is determined, thechronic inflammatory disease may be selected from one or more ofrheumatoid arthritis; osteoarthritis; inflammatory bowel disease;psoriasis; lupus erythematosus; multiple sclerosis; Sjogren's syndrome;polymyositis; dermatomyositis; vasculitis; asthma and mixed connectivetissue disease. When the concentration and/or relative amount of GrA isdetermined, the fibrillinopathy may be selected from one or more of:Marfan syndrome; Beal's syndrome; congenital contractural arachnactyly;supravalvular aortic stenosis; Williams-Beuren syndrome; autosomalrecessive cutis laxa; autosomal dominant cutis laxa; and acquired cutislaxa.

When the concentration and/or relative amount of GrB is determined, thechronic inflammatory disease may be selected from one or more of:selected from one or more of: osteoarthritis; inflammatory boweldisease; psoriasis; lupus erythematosus; scleroderma; multiplesclerosis; polymyositis; dermatomyositis; vasculitis; asthma; and mixedconnective tissue disease. When the concentration and/or relative amountof GrB is determined, the fibrillinopathy may be selected from one ormore of: selected from one or more of: Marfan syndrome; Beal's syndrome;congenital contractural arachnactyly; supravalvular aortic stenosis;Williams-Beuren syndrome; autosomal recessive cutis laxa; autosomaldominant cutis laxa; and acquired cutis laxa.

The fibronectin or fibrillin, may be a fibronectin degradation productor a fibrillin degradation product. The elastin or fibrillin, may be anelastin degradation product or a fibrillin degradation product.

The method may, for example, further include one or more of: diagnosticimaging; clinical diagnosis and alternative laboratory diagnostics.Diagnostic imaging may be selected from one or more of X-ray, computedtomography (CT) scanning, angiography, magnetic resonance imaging (MRI),and ultrasound (for example, see echogenicity vessel plaque assessmentin Skjelland, et al., Atherosclerosis 195:e142-e146 (2007)), dependingon the disease indication and as known in the art. Clinical diagnosismay depend on the disease indication and is well within the skill andknowledge of a person of skill in the art. Alternative laboratorydiagnostics may be selected from any number of known assay methods, butexcluding determining the concentration and/or relative amount of GrAand/or GrB in a blood or serum sample from a subject and comparing theconcentration and/or relative amount to the corresponding concentrationin a control sample, and/or may further exclude identifying one or moreconcentrations and/or relative amounts of one or more of fibrinogen,elastin and/or fibrillin.

“Atherosclerosis” (or “Arteriosclerosis”) in the present context arepotential; causes of coronary artery disease. Atherosclerosis is abuildup of plaque on the inside of arteries. Atherosclerosis ischaracterized by the thickening of the arterial wall, usually at sitesin the arterial tree where laminar flow is disrupted. This inflammatoryvasculopathy is characterized by the excessive accumulation of lipidsand modified lipids is the intima, medial damage, and the thickening andstructural re-organization of the vessel wall. Physical forces, or theexposure to elevated levels of circulating low density lipoprotein (LDL)or free radicals caused by smoking, hypertension, or diabetes mellituscan cause endothelial dysfunction. These factors alter endothelialfunction by increasing the release of pro-inflammatory cytokines andvasoactive substances, which can result in interference with normalanti-thrombotic properties and permeable barrier functions, andincreasing expression of cell-surface adhesion molecules.Atherosclerosis begins as a fatty streak consisting of atherogeniclipoproteins entering the intima and becoming modified. The increase ofcell-surface adhesion molecules causes the recruitment and intravasationof leukocytes, monocytes and T-cells. Pro-inflammatory cytokinesexpressed within the developing lesion provide chemotactic stimulus tothe adherent leukocytes, increasing their migration into the intima.Monocyte colony stimulating factor (M-CSF), which is also produced inthe plaque, augments the expression of macrophage scavenger receptors touptake modified lipids. Macrophages phagocytose this modified lipid inan unregulated manner, causing the formation of foam cells, which makeup the fatty streak. Leukocytes, as well as resident vascular wallcells, secrete cytokines and growth factors that promote the migrationand proliferation of smooth muscle cells (SMC). Vascular SMC (VSMC) mayalso release factors that degrade elastin and collagen in response toinflammatory stimulation, which allows the cells to migrate through theelastic lamina and collagenous matrix. VSMC proliferate and migrate fromthe media to the developing plaque in the intima, and contribute to thefatty streak development into an intermediate lesion by excessiveextracellular matrix (ECM) secretion. This ECM increases the retentionand aggregation of lipoproteins. As the plaque continues to grow,additional lymphocyte recruitment follows, and VSMC form a fibrotic capunder the endothelial layer. The fibrous cap eventually becomes thin andweak by a combination of an inhibition of collagen synthesis from VSMCand the expression of collagenases by foam cells. Eventually, a lesioncan develop that is vulnerable to rupture, exposing thrombogenicmaterial in the form of necrotic foam cells. The plaque may also growwithout rupture, and may eventually obstruct blood flow. The formationof a thrombus which may block blood flow or the obstruction of a vesselfrom plaque formation can lead to ischemia of distal tissue.

“Fibrillinopathies” (type 1) are caused by defects in the fibrillin-1protein (from FBN1 gene). While Marfan syndrome is the most common typeof fibrillinopathy, not all mutations in the FBN1 gene cause thissyndrome. FBN1 mutations cause a spectrum of connective tissuedisorders, with a broad range is severity and age of onset. Some FBN1mutations cause a severe disorder that is fatal to newborns, while othermutations cause adult onset fibrillinopathies with a single abnormality,such as a dislocated lens in the eye or an abnormal aorta.

“Marfan syndrome” (or Marfan's syndrome) is a genetic disorder of theconnective tissue. It is sometimes an inherited dominant trait (carriedby the FBN1 gene). The FBN1 gene encodes a connective protein calledfibrillin-1. The most serious complication is defects of the heartvalves and aorta. It may also affect the lungs, eyes, dural sacsurrounding the spinal cord, skeleton and hard palate.

“Beals syndrome” (or Arachnodactyly, Contractural Beals Type,Beals-Hecht Syndrome, Congenital Contractural Arachnodactyly (CCA)) isan extremely rare genetic disorder caused by a mutation in fibrillin-2gene (FBN2) and characterized by the permanent fixation of certainjoints (e.g., fingers, elbows, knees, and hips) in a flexed position(contractures); abnormally long, slender fingers and toes(arachnodactyly); permanently flexed fingers (camptodactyly); and/orabnormally shaped ears resulting in a “crumpled” appearance. Inaddition, affected individuals may exhibit front-to-back andside-to-side curvature of the spine (kyphoscoliosis); feet that areabnormally positioned (talipes equinovarus or clubfoot); outwarddisplacement of the fingers (ulnar deviation of the fingers); anabnormally short neck; and/or displacement of the lens of the eye(ectopia lentis). In some cases, affected individuals may have a slightdeformity of the valve on the left side of the heart (mitral valveprolapse). Beals syndrome is inherited as an autosomal dominant trait.

“Supravalvular aortic stenosis” (or SVAS) is a fixed form of congenitalleft ventricular outflow tract (LVOT) obstruction that occurs as alocalized or a diffuse narrowing of the ascending aorta beyond thesuperior margin of the sinuses of Valsalva. It accounts for less than 7%of all fixed forms of congenital LVOT obstructive lessons. SVAS mayoccur sporadically, as a manifestation of elastin arteriopathy, or aspart of Williams syndrome.

“Williams syndrome” (or WS, Williams-Beuren syndrome, WBS) is a rareneurodevelopmental disorder caused by a deletion of about 26 genes fromthe long arm of chromosome 7. It is characterized by a distinctive,“elfin” facial appearance, along with a low nasal bridge; an unusuallycheerful demeanor and ease with strangers, coupled with unpredictablyoccurring negative outbursts; a predisposition to violent outbursts;mental retardation coupled with unusual (for persons who are diagnosedas mentally retarded) language skills; a love for music; andcardiovascular problems, such as supravalvular aortic stenosis andtransient hypercalcaemia. The deleted region typically includes CLIP2,ELN, GTF2I, GTF21RD1, and LIMK1. Loss of the ELN gene (elastin), isassociated with the connective-tissue abnormalities and cardiovasculardisease (specifically supravalvular aortic stenosis (SVAS) andsupravalvular pulmonary stenosis (SVPS)) found in many people with thissyndrome.

“Cutis laxa” (or CL) is a rare, inherited or acquired connective tissuedisorder in which the skin becomes inelastic and hangs loosely in folds.The clinical presentation and the mode of inheritance show considerableheterogeneity. Autosomal dominant, autosomal recessive, and X-linkedrecessive patterns have been noted in inherited forms. A serine toproline amino acid substitution in the fibulin 5 (FBLN5) gene has beenassociated with abnormal elastogenesis, resulting in a recessive form ofCL in humans. The X-linked form is currently classified in the group ofcopper transport diseases. The precise cause is unknown, but it may bedue to abnormal elastin metabolism resulting in markedly reduced dermalelastin content.

Methods for identifying a subject being at risk or having a chronicinflammatory disease, fibrillinopathy atherosclerosis, and/or coronaryartery disease, may be supplemented by, in addition to identifying alevel of granzyme A or B in a first sample from the subject, identifyinga level of fibronectin or a level of elastin or a level of fibrillin ina second sample from the subject, identifying a level of fibronectin ora level of elastin or a level of fibrillin in a second normal samplefrom the normal subject not at risk or having the a chronic inflammatorydisease, fibrillinopathy, atherosclerosis, and/or coronary arterydisease and comparing the level of granzyme A or B in the first samplefrom the subject to the level of granzyme B in the first normal samplefrom the normal subject as well as comparing the level of fibronectin orelastin or fibrillin in the second sample from the subject to the levelof fibronectin or elastin or fibrillin in the second normal sample fromthe normal subject. The subject is more likely to be at risk for orhaving a chronic inflammatory disease, fibrillinopathy, atherosclerosis,and/or coronary artery disease when the level of fibronectin or elastinor fibrillin in the second sample from the subject is lower than thelevel of fibronectin or elastin or fibrillin in the second normal samplefrom the normal subject or the level of fibronectin or elastin orfibrillin fragments are higher in the blood than normal subjects.Granzyme A or B cleaves extracellular matrix proteins and when a subjecthas an elevated or high level of granzyme A or B as described above, thelevel of extracellular matrix proteins in the subject will be reduced bythe action of granzyme A or B and increased fragments may appear in theblood. The longer that high levels of granzyme A or B have been active,the lower the tissue levels of extracellular matrix proteins will be atthe disease sites. Baseline levels in healthy subjects for GrB is <40pg/ml and for GrA is <20 pg/ml. Similarly, about 325 ug/ml offibronectin in the blood is a good baseline in healthy subjects(Stathakis et al., J. Clin. Pathol. 34:504-508 (1981)).

Antibody Production

One methodology is to detect the presence of GrA or GrB or elastin orfibronectin or fibrillin specific peptides or proteins. GrA or GrB orelastin or fibronectin or fibrillin specific peptides or proteins mayalso include degradation products thereof. These peptides or proteinsmay be detected by isolating proteinaceous material from a biologicalsample and determining the sequence of peptides or proteins so isolatedand comparing to the known sequence of GrA or GrB or elastin orfibronectin or fibrillin proteins. Preferably, such detecting will makeuse of an intermediate agent such as an antibody specific for the GrA orGrB or elastin or fibronectin or fibrillin peptide or protein as knownin the art.

Antibodies to GrA or GrB or elastin or fibronectin or fibrillin peptidesor proteins may be prepared by a variety of known methods. Suchantibodies may be polyclonal, monoclonal, or may be fragments ofantibodies.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith a GrA or GrB or elastin or fibronectin or fibrillin peptide orprotein fragment which has immunogenic properties. Depending on the hostspecies, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gels such as aluminum hydroxide, and surface active X substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvantsused in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare especially preferable.

It is preferred that GrA or GrB or elastin or fibronectin or fibrillinor fibrillin peptides, fragments, or oligopeptides used to induceantibodies have an amino acid sequence consisting of at least five aminoacids, and more preferably at least 10 amino acids. It is alsopreferable that they are identical to a portion of the amino acidsequence of the natural protein, and they may contain the entire aminoacid sequence of the GrA or GrB or elastin or fibronectin or fibrillinpeptide or protein. Short stretches of GrA or GrB or elastin orfibronectin or fibrillin amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Peptides corresponding to a GrA or GrB or elastin or fibronectin orfibrillin amino acid sequence may be synthesized using methods known inthe art, including the recombinant techniques disclosed in the examplesbelow. Such peptides may also be made to incorporate a N-terminalcysteine to facilitate conjugation to other molecules (e.g., to enhanceimmunogenicity) with such conjugation being mediated by an agent such asm-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Antibodies thatspecifically react with the peptide may be purified from the antisera byaffinity chromatography, for example by using Cellulofine (SeikagakuCorporation) conjugated with the peptide. The resulting antibodies maybe tested by immunoblotting.

Monoclonal antibodies to GrA or GrB or elastin or fibronectin orfibrillin peptides or proteins or anti-idiotypic monoclonal antibodiesmay be prepared using any technique, which provide for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al.(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030;Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120).

One process for obtaining the hybridomas of this invention involvesstarting from spleen cells of an animal, e.g., mouse or rat, previouslyimmunized in vivo or from spleen cells of such animals previouslyimmunized in vitro with an antigen and fusing the immunized cells withmyeloma cells under hybridoma-forming conditions; and selecting thosehybridomas which secrete the monoclonal antibodies which are capable ofspecifically recognizing the GrA or GrB or elastin or fibronectin orfibrillin peptide or protein.

Selected hybridomas are cultured in appropriate culture medium; and thenthe secreted monoclonal antibodies are recovered; or alternatively theselected hybridoma is implanted into the peritoneum of a mouse and, whenascites has been produced in the animal; the monoclonal antibodiesformed from the ascites are recovered. Monoclonal antibodies of theinvention may be prepared by conventional in vitro techniques such asthe culturing of immobilized cells using, e.g., hollow fibers ormicrocapsules or such as the culturing of cells in homogeneoussuspension using, e.g., airlift reactors or stirred bioreactors.

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceGrA or GrB or elastin or fibronectin or fibrillin-specific single chainantibodies. Antibodies with related specificity, but of distinctidiotypic composition, may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton D. R. (1991) Proc. Natl.Acad. Sci. 88:11120-3). Such single chain antibodies may also be usedfor production anti-idiotypic antibodies for use in this invention.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites specific for GrAor GrB or elastin or fibronectin or fibrillin peptides or proteins orfor anti-GrA or GrB or elastin or fibronectin or fibrillin antibodiesmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively Fab expression libraries may be constructed to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity (Huse W. D. et al. (1989) Science 254:1275-1281). Suchfragments when specific for anti-GrA or GrB or elastin or fibronectin orfibrillin antibodies may be used for production of anti-idiotypicantibodies or fragments thereof.

Monoclonal antibodies of this invention may be “chimeric”, an example ofwhich is an animal antigen-binding variable domain coupled to a humanconstant domain (Cabilly et al., U.S. Pat. No. 4,856,567; Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne G.L. et al., Nature 312:643-646 (1984); Neuberger M. S. et al., Nature314:268-270 (1985)). The term “chimeric” antibody describes apolypeptide comprising at least the antigen binding portion of anantibody molecule linked to at least part of another protein such as animmunoglobulin constant domain. However, antibodies of this inventionmay be conjugated to a variety of moieties including labeling moieties.

Various immunoassays may be used for screening to identify antibodieshaving a desired specificity. Numerous protocols for competitive bindingor immunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.Such immunoassays typically involve the measurement of complex formationbetween a GrA or GrB or elastin or fibronectin or fibrillin antigen andits specific antibody. Monoclonal-based immunoassays utilizingmonoclonal antibodies reactive to at least two non-interfering epitopesare preferred, but competitive binding assays may also be employed(Maddox D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

Antibody Assay Methods

One of the most important utilities of the antibodies andproteins/peptides of the invention is for diagnostic purposes, inparticular in assays to detect of quantify the presence of GrA or GrB orelastin or fibronectin or fibrillin antibodies or antigen (GrA or GrB orelastin or fibronectin or fibrillin protein or peptide) in a sample. Inthe following, such assays, in particular ELISAS (enzyme-linkedimmunosorbent assays) and Western blots can be used to detect GrA or GrBor elastin or fibronectin or fibrillin proteins or peptides in samples.Numerous immunoassays are known in the art (Methods in Cell Biology,Vol. 37: Antibodies in Cell Biology, Asai, ed., Academic Press, Inc.,New York (1993); and Basic and Clinical Immunology, 7^(th) ed., a Stites& Terr, eds. (1991)).

A preferred method for detecting GrA or GrB or elastin or fibronectin orfibrillin proteins is the ELISA, in which an antibody typically is boundto an enzyme, such as peroxidase or phosphatase, which can producecolored reaction products from an appropriate buffer. Thus, it utilizesa tagged antigen molecule of known quantity to determine an unlabeledantigen of unknown quantity. Preferably, a GrA or GrB or elastin orfibronectin or fibrillin protein according to the invention, or asuitable functional fragment thereof, is used coupled to a conventionaltag, such as His6.

Thus, in an ELISA format according to the invention, polypeptides orproteins specific for GrA or GrB or elastin or fibronectin or fibrillinare detected and/or quantified, preferably in a biological sample. Thesample may be any sample of biological tissue or fluid, such as blood.The sample is pretreated as necessary by dilution in a suitable buffersolution or concentrated, if desired. Any number of standard aqueousbuffer solutions may be used, such as Tris or the like, at physiologicalpH. Samples are incubated with an excess of the protein according to theinvention as antigen. After rinsing to remove any unbound antigen, theamount of bound antigen is quantitated by adding a solution ofenzyme-conjugated antibody that binds to constant domains of antibodiesin the sample. Excess conjugated antibody is rinsed away and theactivity of the bound enzyme is determined by adding the substrate tothe reaction and measuring the formation of products. As the products ofthe reactions used in ELISA procedures are colored, the amount ofproduct formed can readily be determined by the intensity of the colourthat has developed using a spectrophotometer. The activity of the boundenzyme is proportional to the amount of antigen-binding antibody in thesample; therefore, the original concentration of such antibodies can beestimated from a series of control assays employing known concentrationsof specific antigens. Similarly, antibodies to GrA or GrB or elastin orfibronectin or fibrillin can be detected in a biological sample usingbound antigen (GrA or GrB or elastin or fibronectin or fibrillin proteinor peptide).

As an alternative method for detecting GrA or GrB or elastin orfibronectin or fibrillin proteins Western blots can be utilized takingadvantage of the GrA or GrB or elastin or fibronectin or fibrillinspecific antibodies described above. Biologic samples containingproteins can be assayed by fractionation on polyacrylamide gels underdenaturing conditions. Alternatively, tris tricine polyacrylamide gelelectrophoresis can be used for improved separation of small peptides inthe range from 1 to 100 kDa (Schägger H. and von Jagow G. (1987)Analytical Biochemistry 166:368-379, and Klafki H-W. et al. (1996)Analytical Biochemistry 237:24-29). The proteins separated in the gelscan then be transferred to a membrane using a variety of methods knownin the art. Membranes can then be probed using GrA or GrB or elastin orfibronectin or fibrillin specific antibodies in a Western blot toidentify the proteins of interest in the biological sample preparations.Numerous Western blotting methods are known in the art (ECL Westernblotting protocol—Amersham; Hsu S. M., Methods Enzymol (1990)184:357-63; Leong M. M. and Fox G. R., Methods Enzymol (1990)184:442-51). Immunodiagnostic methods for granzymes A and B are alsodescribed in WO 99/54737.

Western Blotting

As an alternative method for detecting GrA or GrB or elastin orfibronectin or fibrillin proteins or peptides Western blots can beutilized taking advantage of the GrA or GrB or elastin or fibronectin orfibrillin specific antibodies described above. Biologic samplescontaining proteins can be assayed by fractionation on polyacrylamidegels under denaturing conditions. Alternatively, tris tricinepolyacrylamide gel electrophoresis can be used for improved separationof small peptides in the range from 1 to 100 kDa (Schägger H. and vonJagow G. (1987) Analytical Biochemistry 166:368-379, and Klafki H-W. etal. (1996) Analytical Biochemistry 237:24-29). The proteins separated inthe gels can then be transferred to a membrane using a variety ofmethods known in the art. Membranes can then be probed using GrA or GrBor elastin or fibronectin or fibrillin specific antibodies in a Westernblot to identify the proteins or peptides or degradation productsthereof of interest in the biological sample preparations. NumerousWestern blotting methods are known in the art (ECL Western blottingprotocol-Amersham; Hsu S. M., Methods Enzymol (1990) 184:357-63; LeonyM. M. and Fox G. R., Methods Enzymol (1990) 184:442-51).

Alternatively GrA or GrB enzyme-linked immunosorbent spot(ELISPOT—Czerkinsky C. et al. (1983) J Immunol Methods 65 (1-2):109-21),dot blots or other proteomic approaches known in the art.

Various alternative embodiments and examples of the invention aredescribed herein. These embodiments and examples are illustrative andshould not be construed as limiting the scope of the invention.

EXAMPLES Methods and Materials

Mice

All animal protocols were approved by the University of British Columbia(UBC) Animal Care Committee. C57Bl/6 mice, C57Bl/6-ApoE−/− andC57Bl/6-GrB−/− mice were obtained from Jackson Laboratories™ (BarHarbor, Me.) (Piedrahita et al., 1992, Proc Natl. Acad Sci 89:4471-4475;Heusel et al., 1994, Cell 76:977-987). The C57Bl/6-ApoE−/− x GrB−/−double knockout (ApoE/GrB-DKO) mice were generated by crossing theC57Bl/6-ApoE−/− and C57Bl/6-GrB−/− mouse strains. Genotyping of the micewas performed using primers and PCR reactions designed for genotypingthese lines from Jackson Laboratories™ (GrB primers: 5′-TGAAG ATCCTCCTGC TACTG C-3′ (SEQ ID NO: 1) and 5′-TCCTG AGAAA GACCT CTGCC-3′ (SEQID NO: 2); ApoE primers: 5′-GCCTA GCCGA GGGAG AGCCG-3′ (SEQ ID NO: 3)and 5′-TGTGA CTTGG GAGCT CTGCA GC-3′ (SEQ ID NO: 4)). The pups wereweaned at 3 weeks of age and then maintained on a 12-hour day and nightcycle with food and water provided ad libitum. At 6-8 weeks of age, micewere maintained on either regular chow or a Western high fat diet(Harian Teklad™) for 30 weeks and were sacrificed to collect blood andtissues.

Tissue and Blood Collection

Animals were overdosed with 2.5% Avertin™ (Sigma™) and perfusion fixedwith four mL of 4% formalin (Sigma™) at a flow rate of 2 mL/min. Thehearts were then rapidly removed, and aortic root sections wereOCT-embedded. Skin samples taken from the back were either OCT-embedded(Tissue-Tek™) or immersion-fixed in 10% formalin for 24 h before beingembedded in paraffin. Blood extracted by cardiac puncture was collectedin EDTA microvette tubes (Sarstedt™), spun at 10,000×g for 7 minutes at4° C., and the separated serum stored at −80° C. until required foranalysis.

Hair Removal

The animal was deeply anesthetized with 2.5% avertin (Sigma) and fur wasremoved with 10 ml of chemical hair remover (NAIR™, Church and DwightCo.) distributed evenly with a swab. After 15 minutes of incubating, theNAIR™ and fur was removed with warm water and dried with paper tissues.

Immunofluorescence

Immunofluorescence was performed on OCT-embedded frozen sections.Briefly, sections were fixed with acetone for 10 min. Backgroundstaining was blocked by incubation of sections with Dako™ protein block(Dako Cytomation™) for 20 minutes then incubation in 10% donkey serumfor 1 hour. Sections were incubated in goat anti-granzyme B (SantaCruz™, 1:50) and rat anti-mouse macrophage/monocyte (Chemicon, 1:50) at4° C. overnight, followed by incubation in donkey anti-goat IgG (AlexaFluor™ 594, 1:500) and donkey anti-rat IgG (Alexa Fluor™ 488, 1:500) for30 min at room temperature in the dark. Slides were mounted withVECTASHIELD™ Hard-set mounting medium with DAPI (Vector Laboratories™,Burlingame, Calif.). Confocal microscopy wax performed using a LeicaAOBS™ SP2 confocal microscope.

Histological Assessment and Quantitation

Serial 10 μm sections of the aortic roots isolated as described stainedwith hematoxylin & eosin, Movat's pentachrome, elastic van Gieson or OilRed 0. ImageProPlus™ (MediaCybernetics™, Silver Spring, Md.) was used toquantify the lesion area per cross section in ten to twenty sections permouse which were dress averaged to provide mean lesion area per mouse.

Statistics

An ANOVA test was performed to determine statistical differences betweenmultiple groups. Statistical differences between two groups weredetermined using a Student's t-test. For both tests, a p value (alphaerror) of 0.05 or less was considered significant.

Example 1 ApoE/Granzyme B Double Knock-Out Mice

Four groups of mice consisting of (1) C57Bl/6 wild-type, (2) C57/ApoE−/− (ApoE-KO), (3) C57/GrB −/− (GrB-KO), and (4) C57 GrB/ApoE-DKO werefed a normal chow or high fat ‘Western’ diet (21% fat, 0.2% cholesterol)for 30 weeks. No obvious phenotypic differences were observed in thesemice during the first 3 months. Mice were sacrificed and tissuesharvested at 30 weeks of age (ApoE KO mice on the Western diet aresacrificed around this age for humane reasons). As reported in theliterature, the ApoE-KO mice had developed severe skin xanthomatosis,hair loss, hair discoloration and numerous atherosclerotic lesions.Surprisingly, the GrB/ApoE-DKO mice demonstrated a significant reductionin both frequency and size of atherosclerotic lesions (FIG. 4).Atherosclerotic lesions in the ApoE/GrB DKO mice decreased in size toless than 15% of the aortic root area, from more than 40% in the ApoE KOmice fed a Western diet.

Interestingly, this difference in atherosclerotic lesions is not due toa change in blood cholesterol or lipoprotein levels, as there is nodifference between the ApoE KO and the ApoE/GrB DKO mice (FIGS. 1 &2)—both total cholesterol and LDL-C plasma concentrations are the same.No significant differences in HDL, LDL and triglycerides are observedbetween ApoE KO (hatched bars) and ApoE/GrB DKO (checked bars) mice feda Western diet (FIG. 3). Removal of granzyme B activity alone (whitebars) does not have a significant effect on the blood lipid profilescompared to the C57/BL6 (black bar).

At 30 weeks, the DKO mice had no visible xanthomas (Table 1). The DKOmice have smooth and unwrinkled skin. The difference in the incidence ofxanthomatosis was surprising, as no previous link between granzyme B andxanthomatosis had been previously identified.

TABLE 1 Cutaneous xanthomatosis is abolished in the absence of granzymeB activity. Genotype Xanthomatosis present C57  0/8 mice GrB- KO 0/10mice ApoE-KO 9/11 mice ApoE/GrB DKO  0/9 mice

The fur in the ApoE mice is patchy, discoloured (graying) and heldweakly in the skin (easily removed by depilatory), while surprisingly,the ApoE/GrB DKO mice retain their dark fur and does not discolour, andis held firmly in the skin—even more so than the granzyme B KO mice. Thehair follicles in the GrB KO and the ApoE/GrB DKO mice are more abundantand embedded deeper in the fatty layer of the skin, compared to thewild-type or the ApoE KO mice (Table 2). A standard Nair-mediated hairremoval procedure takes more than 45 minutes in the GrB KO and ApoE/GrBDKO mice, compared to 5 minutes in the wild-type or ApoE KO mice.

TABLE 2 Hair follicle density and distribution - Values indicate numberof follicles per ~8.9 mm². N = 2 for each strain Mouse Strain Epidermisand Dermis Sub-dermis C57/B16 12 10 GrB KO 25 6 ApoE KO 12 2 ApoE/GrBDKO 40 36

ApoE KO mice exhibit signs of premature aging, necessitating sacrificeby about 30 weeks (6-7 months) of age, however, the ApoE/GrB DKO miceremain healthy and vigorous beyond 12 months of age, with no visiblesigns of aging or illness. This was surprising, as no support orindication of a role for GrB in longevity has been previously identifiedin the literature.

Co-localization of granzyme B and macrophages in the lesions of theaortic roots were performed and imaged by confocal microscopy. The ApoE−/− lesions showed both granzyme B and macrophage staining, however,co-localization of both occurred at specific regions of the plaque: thefibrotic cap and the shoulder regions. Granzyme B staining was localizedat the internal elastic lamina.

Example 2 Elastin and Granzyme B Distribution in Aortic Sections

Colocalization of granzyme B and macrophages in the lesions of theaortic roots were performed and imaged by confocal microscopy. Thelesion of the ApoE-KO mice showed both granzyme B and macrophagestaining, however, colocalization of both occurred at specific regionsof the plaque: the fibrotic cap and the shoulder regions. Granzyme Bstaining was localized to the elastic lamellae.

In order to adhere to the aortic walls, smooth muscle cells requireelastin. Aortas of C57 wt, GrB −/−, ApoE −/− and DKO mice were stainedwith elastic van Gieson (FIG. 5A to 5D). The aortic wall of the ApoEmouse is very thin and elastin staining is markedly reduced compared tothe C57 wt. In the DKO mouse, the aorta wall is significantly thickerand elastin staining is correspondingly more intense. GrB alsocolocalizes with the internal elastic lamina of atherosclerotic plaquesand an influx of macrophages in the ApoE −/−. Surprisingly, thiscolocalization is not observed in the DKO mice, as demonstrated byconfocal microscopy staining

The increased localization of granzyme B with the internal elasticlamina indicates that it may accumulate on elastin fibres and over time,contribute to degradation of elastin. This in turn would lead to reducedelasticity, production of fragments that enhance inflammation, increasedcalcification and overall stiffness (hardening) of blood vessels.Reduced elastin in the internal elastic lamina also promotes migrationof smooth muscle cells in to the intima (intimal hyperplasia) and theformation of atherosclerotic plaques. The fragmented and degradedelastin (by granzyme B) may lead to recruitment of immune cells of thelesion.

Example 3 Reduced Cutaneous Inflammation in DKO Mice

The skin of ApoE −/− mice appears much more aged, unhealthy and is veryfragile. The skin has markedly reduced elasticity, which is restored inthe DKO mice, where granzyme B activity is absent (FIG. 6).

In FIG. 7, an area of massive immune cell infiltration in the ApoE −/−mice (circled area) is visible, that is not observed in the DKO mice.

This immune cell recruitment and inflammatory response may be aconsequence of the chemotactic action of the cleaved extracellularmatrix components such as elastin fragments and fibronectin fragments.The presence of granzyme B in the ApoE −/− mice localizes to these sitesand may be contributing to the generation of these fragments. Thisinflammatory effect (and granzyme B localization) is not observed in theDKO mice.

Additionally, granzyme B may contribute to matrix degradation and/orremodeling of matrix composition, as areas are ‘lost’ or left unstainedin the fixation process of tissues from GrB −/− or DKO mice. Granzyme Bmediated degradation of matrix, in the presence of high lipids (asobserved in the ApoE −/− mice) may contribute to the phenotype observed.

Example 4 Granzyme B Binds to the Extracellular Matrix Protein Elastin

An in vitro granzyme B elastin binding assay was conducted in thefollowing manner. Granzyme B at 50, 100 and 300 ng was incubated with 15μg of human insoluble skin (Sk) and aortic (Ao) elastin (ElastinProducts Company Owensville, Mo.) in PBS for three hours at roomtemperature. The samples were centrifuged at 1000×g at room temperaturefor three minutes and the insoluble elastin collected in the pellet. Thesupernatants, which contained unbound granzyme B, were denatured withSDS loading buffer and run on a 10% SDS-PAGE gel. Granzyme B wasdetected by Western blot. Each gel contained three lanes: a first lanerelated to a sample containing granzyme B in the absence of elastin; asecond lane related to the samples containing granzyme B and humaninsoluble skin elastin; and a third lane related to the samplecontaining granzyme B and aortic elastin. The lane relating to thesample containing granzyme B in the absence of elastin showed a heavyband in the supernatant and a faint band in the pellet. The lanesrelating to the samples containing granzyme B and skin elastin, andgranzyme B and aortic elastin both showed heavy bands in the pellet,which bands were much heavier than the faint band seen in the pelletrelating to the sample containing granzyme B in the absence of elastin.Furthermore, the band in the supernatant for the sample containinggranzyme B and skin elastin was dramatically less pronounced than thesupernatant band shows in the sample relating to granzyme B in theabsence of elastin. No band appeared in the supernatant samplecontaining granzyme B and aortic elastin. Hence, there is less granzymeB present in the supernatant, thus indicating that granzyme B wasassociating with the elastin in the pellet. This phenomenon wasdose-dependent and not restricted to the type of elastin used (i.e.,skin elastin or aortic elastin).

Example 5 Granzyme B Cleaves Extracellular Matrix Proteins

Treatment of human coronary artery smooth muscle cells (SMC) matrix withgranzyme B induced a cleavage of a number of extracellular proteins.Extracellular proteins from SMC cultures were biotinylated and incubatedwith granzyme B. The supernatant was collected at 2, 4 and 24 hoursafter treatment, and the entire insoluble extracellular proteinpreparation collected at 24 hours. Extracellular proteins werevisualized by Western blot for biotin. Western blot for beta-actinconfirmed that the extracellular protein preparation was devoid ofintercellular proteins. Western blots for fibronectin, phosphorylatedFAK (p-FAK), and FAK were also performed on lysates of SMC treated withgranzyme B. In the collected insoluble proteins, four protein bandsbetween approximately 50-70 kDa and approximately 236 kDa disappeared 24hours after treatment with granzyme B and cleavage of fragmentsapproximately 25-39 kDa were evident in the matrix at this same timepoint. Further, the six proteins and/or cleavage fragments ranging inmolecular weight from approximately 29-148 kDa were eluted into thesupernatant as early as two hours after granzyme B treatment. To ensurethat the SMC extracellular protein preparations used were devoid ofintracellular proteins, Western blotting for beta-actin was performed onthe collected supernatant and extracellular proteins. Beta-actin wasapparent in SMC lysates (positive control) but was absent from matrixand supernatant preparations.

To identify extracellular proteins that are cleaved by granzyme B,Western blots for fibronectin, collagen, and vitronectin on lysates fromuntreated and granzyme B-treated SMCs were performed. In all SMCstreated with granzyme B for 24 hours, there was a reduction in the totalamount of fibronectin in lysates collected from SMCs. In thesupernatants of granzyme B-treated SMCs at 24 hours, a fibronectincleavage product was detected. There was no cleavage of collagen IV orvitronectin was observed. Therefore, granzyme B induces a cleavage offibronectin in SMC extracellular matrixes but does not affect collagenIV or vitronectin.

Also human coronary artery smooth muscle cells were cultured toconfluency and serum starved for 48 hours at which time cells were lysedwith NH₄OH so that the intact extracellular matrix (ECM) remained on theplate. Granzyme B (80 nm) was incubated on the ECM for 24 hours at roomtemperature. Supernatants (containing cleaved ECM) and ECM stillattached to the plate were collected and assessed for fibrillin cleavageby Western blot. The results, not shown, may be summarized as follows:Western blots of PBS (negative control), Trypsin (positive control) andGrB supernatants and PBS, Trypsin and GrB ECMs were performed with afibrillin-1 antibody, which showed fibrillin-1 cleavage fragments in theGrB supernatant, GrB ECM, Trypsin supernatant and Trypsin ECM, but notin the PBS supernatant or ECM. Six independent experiments were carriedout and three representative groups were tested. Results confirm thatGrB cleaves fibrillin-1 in human coronary artery smooth muscle cells.

Example 6 Granzyme B Binds and Degrades Elastin In Vitro

Tritiated elastin was prepared with the modifications as described inBanda M. J. and Werb Z. (1981) Biochem J 193:589-605, and Gordon, S.,Werb, Z. and Cohn Z. A. (1976) in In Vitro Methods in Cell Mediated andTumor Immunity, eds. Bloom B. R. and David J. R. (Academic Press, NewYork), pages 349-350. 1 mg of skin or aortic elastin was diluted in 1 mldH₂O and pHed to 9.2. 1 mCi NaB₃H₄ (PerkinElmer, Waltham, Mass.) and 2mg of non-radioactive NaB₃H₄ (Sigma, St. Louis, Mo.) was added. After 2hours of incubation, the pH was adjusted to 3.0 and the elastin wasincubated for an additional 30 minutes. The elastin was centrifuged for3 minutes at 5000×g and the pellet was repeatedly washed to removeexcess NaB₃H₄. For the cleavage assays, 0.15 mg ³H-elastin was incubatedwith granzyme B (0.75 μg was added a total of 5 times) at roomtemperature for 7 days. At day 7 of incubation, 25 μg of elastase(Elastin Products Company, Owensville, Mo.) was incubated with elastinfor 2 hours, as a positive control. After incubations, reactions werecentrifuged at 5000×g for 3 minutes. The radioactivity of the soluble,cleaved elastin fragments in the supernatant was counted in Ready SafeScintillation Fluid (Beckman-Coulter, Fullerton, Calif.). Theradioactivity of the cleaved, soluble elastin fragments was 4.8 timesand 2.7 times higher than background for skin and aortic elastin,respectively (FIG. 11). Proteolysis of elastin by elastase yielded aradioactivity increase over background of 14.9 fold for skin elastin and7.7 fold for aortic elastin. These data show that granzyme B hasaffinity to elastin and has elastolytic activity.

Example 7 Whole Blood Plasma Protocol

7.5 ml blood samples were collected from normal subjects (GOLD 1 & 2)and from subjects having chronic obstructive pulmonary disease (GOLD 3 &4) using a purple top EDTA vacutainer tube (BD). Immediately uponcollection, the tube was inverted 5 times for thorough mixing. The tubeswere then centrifuged for 11 min at 276×g (Beckman Coulter). Followingcentrifugation, the tubes are separated into 3 distinct layers: a bottomlayer of mostly red blood cells, a thin film layer of white blood cells(buffy coat) and a top layer of plasma. Using a sterile transferpipette, the top layer of plasma down to about 1 mm from the red bloodcells was removed, being careful not to aspirate the buffy coat, and theplasma was placed into a labeled orange top cryotube. The samples werestored immediately at −80° C. until plasma analysis was performed.

Granzyme B Analyses Using ELISA Kits from Bender Medsystems

For plasma analysis, human Granzyme B ELISA kits were used (catalognumber: BMS2027). The kits comprise enzyme-linked immunosorbent assayfor quantitative detection of human granzyme B. The reagents wereprepared as per the kit's protocols: a) Wash Buffer; b) Dilution Buffer;c) Biotin-Conjugate; d) Granzyme standards; e) Streptavidin-HRP; and f)Colour-giving reagents: Blue-Dye, Green-Dye, Red-Dye.

The assays were performed as per the kit's protocols and the calculationof the results were performed as per the kit's protocols. The resultsare depicted graphically in FIGS. 8, 9, and 10 and in Table 3 below.

TABLE 3 Granzyme B in the lung GRB GRB/Protein [pg/ml] [pgGRB/mgProtein]WL COPD 4090.47 514.93 (N = 3) Control 1201.94 212.36 (n = 2) Cyt COPD3559.18 346.66 (N = 3) Control 1072.52 140.57 (n = 2) M COPD 57.33 58.77(n = 3) Control 7.80 8.26 (n = 2)Normal (Control) or COPD-afflicted lungsections were isolated. Proteinlevels of Granzyme B in whole lysates (WL), cytosol (Cyt), or microsomes(M) were assessed using ELISA.

Example 8 Whole Blood Plasma Protocol

7.5 ml blood samples were collected from normal subjects and fromsubjects having chronic obstructive pulmonary disease using a purple topEDTA vacutainer tube (BD). Immediately upon collection, the tube wasinverted 5 times for thorough mixing. The tubes were then centrifugedfor 11 min at 276×g (Beckman Coulter). Following centrifugation, thetubes are separated into 3 distinct layers: a bottom layer of mostly redblood cells, a thin film layer of white blood cells (buffy coat) and atop layer of plasma. Using a sterile transfer pipette, the top layer ofplasma down to about 1 mm from the red blood cells was removed, beingcareful not to aspirate the buffy coat, and the plasma was placed into alabeled orange top cryotube. The samples were stored immediately at −80°C. until plasma analysis was performed.

Granzyme A Analyses Using ELISA Kits from Bender Medsystems

For plasma analysis, human Granzyme A ELISA kits were used (catalognumber: BMS2026). The kits comprise enzyme-linked immunosorbent assayfor quantitative detection of human granzyme A. The reagents wereprepared as per the kit's protocols: a) Wash Buffer; b) Dilution Buffer;c) Biotin-Conjugate; d) Granzyme standards; e) Streptavidin-HRP; f) TMBsubstrate solution; and g) Colour-giving reagents: Blue-Dye, Green-Dye,Red-Dye.

The assays were performed as per the kit's protocols and the calculationof the results was performed as per the kit's protocols. The results aredepicted graphically in FIGS. 12 and 13. FIG. 12 illustrates the resultswhereby granzyme A is elevated in the plasma of patients with confirmedatherosclerosis. Plasma samples were obtained from patients with (n=5)or without (n=5) coronary artery disease (CAD) from the Healthy HeartClinic at St. Paul's Hospital in Vancouver, British Columbia, Canada.CAD was determined by angiography and granzyme A levels were measured asdescribed above. FIG. 13 illustrates the results whereby granzyme Alevels are elevated in the plasma of patients that smoke. Plasma sampleswere obtained from patients that do (n=3) or do not (n=3) smoke. Sampleswere obtained from the Health Heart Clinic at St. Paul's Hospital, inVancouver, British Columbia, Canada. Plasma levels of granzyme A weremeasured as described above.

Example 9

Granzyme A-mediated proteolysis of smooth muscle cell (SMC)-generatedextracellular proteins is illustrated in FIGS. 14A and 14B. SMC werecultured until confluency and then lysed with 0.25 M NH₄OH for 30 min.The remaining extracellular matrix proteins were biotinylated andincubated with granzyme A. Supernatants were collected at 1, 2, 6, 16and 24 h post-treatment. FIG. 14A illustrates the results wherebyextracellular proteins were visualized by Western blot for biotin.Increased levels of fragmented extracellular matrix proteins wereobserved in granzyme A-treated plates. FIG. 14B illustrates the resultswhereby supernatants treated with granzyme A for the indicated timeswere probed for fibronectin and several fragments were observed asindicated by the arrows.

Granzyme A cleaves fibrillin-1 in vitro. Human coronary artery smoothmuscle cells were cultured to confluency and serum starved for 48 hoursat which time cells were lysed with NH₄OH so that the intactextracellular matrix (ECM) remained on the plate. Granzyme A (100 nM) inPBS was incubated on the ECM for 24 hours at room temperature.Supernatants were collected and assessed for fibrillin presence and sizeby SDS-PAGE and subsequent fibrillin-1 Western blot (results not shown).The results, may be summarized as follows: Western blots of PBS(negative control), Trypsin (positive control) and GrA supernatants andPBS, Trypsin and GrA ECMs were performed with a fibrillin-1 antibody,which showed fibrillin-1 cleavage fragments in the GrA supernatant, GrAECM, Trypsin supernatant, and Trypsin ECM, but not in the PBSsupernatant or ECM. Six independent experiments were carried out and 3representative groups were tested. Results confirm that GrA cleavesfibrillin-1 in human coronary artery smooth muscle cells. Furthermore,GrB has also been shown to cleave Fibrillin-2 and Fibulin-2 in humancoronary artery smooth muscle cells (HCASMC)-derived ECM (data notshown) and GrB cleavage is attenuated by the granzyme B inhibitordichloroisocoumarin (DCI).

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of diagnosis ofatherosclerosis or coronary artery disease in a subject suspected ofhaving atherosclerosis or coronary artery disease or havingatherosclerosis or coronary artery disease, the method comprising: a)determining a concentration of Granzyme B (GrB) in a blood plasma orserum sample from said subject; and b) comparing said concentration to acorresponding concentration in a control sample, wherein an elevatedconcentration of GrB is indicative of atherosclerosis or coronary arterydisease.
 2. The method of claim 1, further comprising determining aconcentration of one or more of: fibronectin and fibrillin; withreference to said control sample as indicative of atherosclerosis orcoronary artery disease.
 3. The method of claim 1, wherein theconcentration of GrB is determined by an immunodiagnostic assay.
 4. Themethod of claim 2, wherein the concentration of fibronectin or fibrillinis determined by immunodiagnostic assay.
 5. The method of claim 3,wherein the immunodiagnostic assay is an enzyme-linked immunosorbentassay (ELISA).
 6. The method of claim 1, wherein the subject has a GrBblood plasma or serum concentration of >40 pg/ml.
 7. A method ofdiagnosis of chronic inflammatory disease in a subject suspected ofhaving chronic inflammatory disease or having chronic inflammatorydisease, the method comprising: a) determining a concentration ofGranzyme A (GrA) in a blood plasma or serum sample from said subject;and b) comparing said concentration to a corresponding concentration ina control sample, wherein an elevated concentration of GrA is indicativeof chronic inflammatory disease.
 8. The method of claim 7, furthercomprising determining a concentration of one or more of: fibronectinand fibrillin; with reference to said control sample as indicative ofchronic inflammatory disease.
 9. The method of claim 7, wherein theconcentration of GrA is determined by an immunodiagnostic assay.
 10. Themethod of claim 8, wherein the concentration of fibronectin or fibrillinis determined by immunodiagnostic assay.
 11. The method of claim 9,wherein the immunodiagnostic assay is an enzyme-linked immunosorbentassay (ELISA).
 12. The method of claim 7, wherein the chronicinflammatory disease is selected from one or more of: rheumatoidarthritis, osteoarthritis, inflammatory bowel disease, psoriasis, lupuserythematosus, multiple sclerosis, Sjogern's syndrome, polymyositis,dermatomyositis, vasculitis, asthma and mixed connective tissue disease.13. The method of claim 7, wherein the subject has a GrA blood plasmaconcentration of >20 pg/ml or a fibronectin blood plasma concentrationof >400 μg/ml.
 14. A method of diagnosis of a fibrillinopathy in asubject suspected of having fibrillinopathy or having fibrillinopathy,the method comprising: a) determining a concentration of (Granzyme A)GrA in a blood plasma or serum sample from said subject; and b)comparing said concentration to a corresponding concentration in acontrol sample, wherein an elevated concentration of GrA is indicativeof fibrillinopathy.
 15. The method of claim 14, further comprisingdetermining a concentration of one or more of: fibronectin andfibrillin; with reference to said control sample as indicative offibrillinopathy.
 16. The method of claim 14, wherein the concentrationof GrA is determined by immunodiagnostic assay.
 17. The method of claim14, wherein the concentration of fibronectin or fibrillin is determinedby immunodiagnostic assay.
 18. The method of claim 16, wherein theimmunodiagnostic assay is an enzyme-linked immunosorbent assay (ELISA).19. The method of claim 14, wherein the fibrillinopathy is selected fromone or more of: Marfan syndrome, Beal's Syndrome, congenitalcontractural arachnactyly, supravalvular aortic stenosis,Williams-Beuren syndrome, autosomal recessive cutis laxa, autosomaldominant cutis laxa, and acquired cutis laxa.
 20. The method of claim14, wherein the subject has a GrA blood plasma concentration of >20pg/ml or a fibronectin blood plasma concentration of >400 μg/ml.
 21. Amethod for diagnosis of chronic inflammatory disease in a subjectsuspected of having chronic inflammatory disease or having chronicinflammatory disease, the method comprising: a) determining aconcentration of (Granzyme B) GrB in a blood plasma or serum sample fromsaid subject; and b) comparing said concentration to a correspondingconcentration in a control sample, wherein an elevated concentration ofGrB is indicative of chronic inflammatory disease.
 22. The method ofclaim 21, further comprising determining a concentration of one or moreof: elastin and fibrillin; with reference to said control sample isindicative of chronic inflammatory disease.
 23. The method of claim 21,wherein the concentration of GrB is determined by an immunodiagnosticassay.
 24. The method of claim 22, wherein the concentration of elastinor fibrillin is determined by an immunodiagnostic assay.
 25. The methodof claim 23, wherein the immunodiagnostic assay is an enzyme-linkedimmunosorbent assay (ELISA).
 26. The method of claim 21, wherein thechronic inflammatory disease is selected from one or more of:osteoarthritis, inflammatory bowel disease, psoriasis, lupuserythematosus, scleroderma, multiple sclerosis, polymyositis,dermatomyositis, vasculitis, asthma and mixed connective tissue disease.27. The method of claim 21, wherein the subject has a GrB blood plasmaconcentration of >40 pg/ml.
 28. The method of any one of claims 14,wherein the fibronectin or fibrillin, may be a fibronectin degradationproduct or a fibrillin degradation product.